Production of a Monoclonal Antibody for Histone H2b Isoform H2b3b.

H2b3b is one of the histone H2b isoforms that differs from canonical H2b by five to six amino acids. Previously, we identified H3t as the testis-specific histone H3 variant located in histone cluster 3, which is also the site of H2b3b. In this study, we produced monoclonal antibodies against H2b3b, using the iliac rat lymph node method for rat antibody and the immunochamber method for rabbit antibody. Immunoblot analysis confirmed that our antibodies could specifically discriminate between H2b3b and canonical H2b. Moreover, immunostaining revealed colocalization with a testicular stem cell marker, Plzf, but not with a meiotic marker, Sycp. This indicated that H2b3b is expressed in spermatogenic cells before meiosis. Our monoclonal antibodies enable further studies to reveal specific functions of H2b3b during spermatogenesis. We also hope that the established method will lead to the production of antibodies that can identify other H2b isoforms.

Expression level of neutrophil extracellular traps in peripheral blood of patients with chronic heart failure complicated with venous thrombosis and its clinical significance.

OBJECTIVE: Previous studies have reported that neutrophil extracellular traps (NETs) have been identified to be involved in thrombosis, but the clinical value in chronic heart failure (CHF) patients with venous thrombosis is unclear. This study focused on the expression level of NETs in the peripheral blood of patients with CHF complicated with venous thrombosis and its clinical value. METHODS: 80 patients with CHF were included and divided into 2 groups according to the occurrence of venous thrombosis, and the expression levels of NETs in peripheral venous blood and lesion veins of the patients were detected through fluorescent staining. Myeloperoxidase-DNA (MPO-DNA) and citrullinated histone H3 (CitH3), markers of NETs, were detected by enzyme linked immunosorbent assay kit. The receiver operating characteristic (ROC) curve was used to analyze the value of peripheral venous blood NETs in the diagnosis of venous thrombosis in CHF patients, while the relationship between NETs in peripheral and lesion veins was analyzed by a unitary linear regression model. RESULTS: The results showed that the concentration of NETs, MPO-DNA, and CitH3 in CHF patients combined with venous thrombosis was markedly higher than that in patients without venous thrombosis, and the concentration of NETs, MPO-DNA, and CitH3 in lesion venous blood was notably higher than that in peripheral venous blood. Binary logistics regression analysis showed that NETs in peripheral venous blood were an independent risk factor for venous thrombosis in patients with heart failure. The unitary linear regression model fitted well, indicating a notable positive correlation between NETs concentrations in peripheral and lesion veins. The area under the ROC curve for diagnosing venous thrombosis was 0.85, indicating that peripheral blood NETs concentration levels could effectively predict venous thrombosis in CHF patients. CONCLUSION: The expression level of NETs was high in the peripheral blood of CHF patients combined with venous thrombosis and was the highest in lesion venous blood. NETs levels in peripheral blood had the value of diagnosing venous thrombosis in CHF patients, and the concentrations of NETs in peripheral and lesion veins are markedly positively correlated.

Prognostic importance of mitosis quantification and PHH3 expression in oral epithelial dysplasia.

Oral epithelial dysplasia (OED) is diagnosed and graded using a range of histological features, making grading subjective and challenging. Mitotic counting and phosphohistone-H3 (PHH3) staining have been used for the prognostication of various malignancies; however, their importance in OED remains unexplored. This study conducts a quantitative analysis of mitotic activity in OED using both haematoxylin and eosin (H&E)-stained slides and immunohistochemical (IHC) staining for PHH3. Specifically, the diagnostic and prognostic importance of mitotic number, mitotic type and intra-epithelial location is evaluated. Whole slide images (WSI) of OED (n = 60) and non-dysplastic tissue (n = 8) were prepared for analysis. Five-year follow-up data was collected. The total number of mitosis (TNOM), mitosis type and intra-epithelial location was manually evaluated on H&E images and a digital mitotic count performed on PHH3-stained WSI. Statistical associations between these features and OED grade, malignant transformation and OED recurrence were determined. Mitosis count increased with grade severity (H&E: p < 0.005; IHC: p < 0.05), and grade-based differences were seen for mitosis type and location (p < 0.05). The ratio of normal-to-abnormal mitoses was higher in OED (1.61) than control (1.25) and reduced with grade severity. TNOM, type and location were better predictors when combined with histological grading, with the most prognostic models demonstrating an AUROC of 0.81 for transformation and 0.78 for recurrence, exceeding conventional grading. Mitosis quantification and PHH3 staining can be an adjunct to conventional H&E assessment and grading for the prediction of OED prognosis. Validation on larger multicentre cohorts is needed to establish these findings.

Proteomic profiling of serum exosomes reveals acute phase response and promotion of inflammatory and platelet activation pathways in patients with heat stroke.

Background: The pathological mechanism of heat stroke (HS) involves the acute phase response, unbalanced immunological/inflammatory reactions, and coagulation initiation, especially platelet activation. Although exosomes contain proteins involved in these biological processes, their protein cargo levels and potential roles in HS remain unknown. This study explored the serum exosome protein expression patterns after HS and their potential roles in the pathogenesis of HS. Methods: Blood samples were collected from ten patients diagnosed with HS upon admission to the intensive care unit (six with severe HS and four with mild HS). Samples from six healthy volunteers were included as control. Using ultracentrifugation, exosomes were prudently isolated, and their protein contents were profiled using liquid chromatography-tandem mass spectrometry analysis with isobaric tags for relative and absolute quantification-based proteomics. Results: Compared with healthy volunteers, patients with HS showed significant changes in the levels of 33 exosomal proteins (23 upregulated and 10 downregulated). The most upregulated proteins included serum amyloid A-1 (SAA-1), von Willebrand factor (vWF), S100A8, and histone H3. In addition, SAA-1, vWF, platelet membrane glycoprotein, S100A8, and histone H3 were more enriched in the exosomes from patients with severe HS than from those with mild HS. Gene ontology analysis revealed that the HS-modulated exosomal proteins were mostly related to inflammatory response, including the acute-phase response, platelet activation/degranulation, and innate immune response. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment of proteins in the IL-17 signaling pathway, platelet activation, neutrophil extracellular trap formation, Fc epsilon RI signaling pathway, chemokine signaling pathway, and NOD-like receptor signaling pathway, among others. Several serum exosomal proteins, including SAA-1, vWF, and S100A8, which are related to the acute phase, inflammatory response, and platelet activation, were confirmed to be elevated in patients with HS, and were significantly correlated with disease severity, organ dysfunction, and death. Conclusion: Overall, this study explores the potential role of the serum exosomal proteome in the inflammatory response and platelet activation in HS, suggests the pathological mechanisms underlying HS-induced injuries, and recommends reliable exosomal biomarkers for predicting HS prognosis.

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

Quantitative Assessment of Epithelial Proliferation in Rat Mammary Gland Using Artificial Intelligence Independent of Choice of Proliferation Marker.

Epithelial proliferation in the rat mammary gland is recommended in regulatory guidelines as an endpoint for assessment of the in vivo carcinogenic potential of insulin analogues. Epithelial proliferation is traditionally assessed by immunohistochemical staining of a proliferation marker, for example, 5-bromo-2'-deoxyuridine (BrdU) or Ki67, followed by labor-intensive manual counting of positive and negative cells. The aim of this study was to develop and validate an approach for image analysis based on artificial intelligence, which can be used for quantification of proliferation in rat mammary gland, independent of the choice of proliferation marker. Furthermore, the aim was to compare the markers BrdU, Ki67, and phosphorylated histone H3 (PHH3). A sequence of image analysis applications were developed, which allowed for quantification of proliferative activity in the mammary gland epithelium. These endpoints agreed well with manually counted labeling indices, with correlation coefficients in the range ≈0.92-0.93. In addition, all three proliferation markers were significantly correlated and could detect the variation in epithelial proliferation during the estrous cycle. In conclusion, image analysis can be used to quantify epithelial proliferation in the rat mammary gland and thereby replace time-consuming manual counting. Furthermore, BrdU, Ki67, and PHH3 can be used interchangeably to assess proliferation.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

The genetic and epigenetic landscape of the Arabidopsis centromeres.

Centromeres attach chromosomes to spindle microtubules during cell division and, despite this conserved role, show paradoxically rapid evolution and are typified by complex repeats. We used long-read sequencing to generate the Col-CEN Arabidopsis thaliana genome assembly that resolves all five centromeres. The centromeres consist of megabase-scale tandemly repeated satellite arrays, which support CENTROMERE SPECIFIC HISTONE H3 (CENH3) occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites that show the least amount of divergence and occur in higher-order repeats. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization. Centromeric crossover recombination is suppressed, yet low levels of meiotic DNA double-strand breaks occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving through cycles of satellite homogenization and retrotransposon-driven diversification.

CHD1 controls H3.3 incorporation in adult brain chromatin to maintain metabolic homeostasis and normal lifespan.

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.

FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers.

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing ∼4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.

Genomic impact of stress-induced transposable element mobility in Arabidopsis.

Transposable elements (TEs) have long been known to be major contributors to plant evolution, adaptation and crop domestication. Stress-induced TE mobilization is of particular interest because it may result in novel gene regulatory pathways responding to stresses and thereby contribute to stress adaptation. Here, we investigated the genomic impacts of stress induced TE mobilization in wild type Arabidopsis plants. We find that the heat-stress responsive ONSEN TE displays an insertion site preference that is associated with specific chromatin states, especially those rich in H2A.Z histone variant and H3K27me3 histone mark. In order to better understand how novel ONSEN insertions affect the plant's response to heat stress, we carried out an in-depth transcriptomic analysis. We find that in addition to simple gene knockouts, ONSEN can produce a plethora of gene expression changes such as: constitutive activation of gene expression, alternative splicing, acquisition of heat-responsiveness, exonisation and genesis of novel non-coding and antisense RNAs. This report shows how the mobilization of a single TE-family can lead to a rapid rise of its copy number increasing the host's genome size and contribute to a broad range of transcriptomic novelty on which natural selection can then act.

Histone H3 lysine 27 acetylation profile undergoes two global shifts in undernourished children and suggests altered one-carbon metabolism.

BACKGROUND: Stunting is a condition in which a child does not reach their full growth potential due to chronic undernutrition. It arises during the first 2 years of a child's life and is associated with developmental deficiencies and life-long health problems. Current interventions provide some benefit, but new approaches to prevention and treatment grounded in a molecular understanding of stunting are needed. Epigenetic analyses are critical as they can provide insight into how signals from a poor environment lead to changes in cell function. RESULTS: Here we profiled histone H3 acetylation on lysine 27 (H3K27ac) in peripheral blood mononuclear cells (PBMCs) of 18-week-old (n = 14) and 1-year-old children (n = 22) living in an urban slum in Dhaka, Bangladesh. We show that 18-week-old children destined to become stunted have elevated levels of H3K27ac overall, functional analysis of which indicates activation of the immune system and stress response pathways as a primary response to a poor environment with high pathogen load. Conversely, overt stunting at 1-year-of age is associated with globally reduced H3K27ac that is indicative of metabolic rewiring and downregulation of the immune system and DNA repair pathways that are likely secondary responses to chronic exposure to a poor environment with limited nutrients. Among processes altered in 1-year-old children, we identified one-carbon metabolism, the significance of which is supported by integrative analysis with results from histone H3 trimethylation on lysine 4 (H3K4me3). Together, these results suggest altered one-carbon metabolism in this population of stunted children. CONCLUSIONS: The epigenomes of stunted children undergo two global changes in H3K27ac within their first year of life, which are associated with probable initial hyperactive immune responses followed by reduced metabolic capacity. Limitation of one-carbon metabolites may play a key role in the development of stunting. Trial registration ClinicalTrials.gov NCT01375647. Registered 17 June 2011, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01375647 .

Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds.

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94-96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time-effect curves up to 8 h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online ( http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/ ). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

Involvement of Neutrophil Extracellular Traps in Cerebral Arteriovenous Malformations.

BACKGROUND: Cerebral arteriovenous malformations (cAVMs) represent tangles of abnormal vasculature without intervening capillaries. High-pressure vascular channels due to abnormal arterial and venous shunts can lead to rupture. Multiple pathways are involved in the pathobiology of cAVMs including inflammation and genetic factors such as KRAS mutations. Neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs), plays a multifunctional role in infection, inflammation, thrombosis, intracranial aneurysms, and tumor progression. However, the relationship between NETs and the pathobiology of cAVMs remains unknown. We tested whether NETs play a role in the pathobiology of cAVMs. METHODS: We analyzed samples from patients who had undergone surgery for cAVM and immunohistochemically investigated expression of citrullinated histone H3 (CitH3) as a marker of NETs. CitH3 expression was compared among samples from cAVM patients, epilepsy patients, and normal human brain tissue. Expressions of thrombotic and inflammatory markers were also examined immunohistochemically in samples from cAVM patients. RESULTS: Expression of CitH3 derived from neutrophils was observed intravascularly in all cAVM samples but not other samples. Nidi of AVMs showed migration of many Iba-I-positive cells adjacent to the endothelium and endothelial COX2 expression, accompanied by expression of IL-6 and IL-8 in the endothelium and intravascular neutrophils. Unexpectedly, expression of CitH3 was not necessarily localized to the vascular wall and thrombus. CONCLUSIONS: Our results offer the first evidence of intravascular expression of NETs, which might be associated with vascular inflammation in cAVMs.

Does timing matter in radiotherapy of hepatocellular carcinoma? An experimental study in mice.

This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on non-tumoral liver (NTL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA-double strand breaks were analyzed in irradiated and nonirradiated animals by detection of Ki67 and γ-H2AX. Prior to whole animal experiments, organotypic slice cultures were investigated to determine the dosage to be used in whole animal experiments. Irradiation was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on NTL were minimal at ZT20. As compared with NTL, nonirradiated HCC revealed disruption in daily variation and downregulation of all investigated clock genes except Per1. Irradiation affected rhythmic clock gene expression in NTL and HCC at all ZTs except at ZT20 (late activity phase). Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice at which the ratio between efficacy of tumor treatment and toxic side effects was maximal. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man.

Evolution of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients.

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.

Insights into the Molecular Mechanisms of Histone Code Recognition by the BRPF3 Bromodomain.

Bromodomains are evolutionarily conserved reader modules that recognize acetylated lysine residues on the histone tails to facilitate gene transcription. The bromodomain and PHD finger containing protein 3 (BRPF3) is a scaffolding protein that forms a tetrameric complex with HBO1 histone acetyltransferase (HAT) and two other subunits, which is known to regulate the HAT activity and substrate specificity. However, its molecular mechanism, histone ligands, and biological functions remain unknown. Herein, we identify mono- (H4K5ac) and di- (H4K5acK12ac) acetylated histone peptides as novel interacting partners of the BRPF3 bromodomain. Consistent with this, pull-down assays on purified histones from human cells confirm the interaction of BRPF3 bromodomain with acetylated histone H4. Further, MD simulation studies highlight the binding mode of acetyllysine (Kac) and the stability of bromodomain-histone peptide complexes. Collectively, our findings provide a key insight into how histone targets of the BRPF3 bromodomain direct the recruitment of HBO1 complex to chromatin for downstream transcriptional regulation.

Assessment of H3K27me3 immunohistochemistry and combination of NF1 and p16 deletions by fluorescence in situ hybridization in the differential diagnosis of malignant peripheral nerve sheath tumor and its histological mimics.

BACKGROUND: A definitive diagnosis of malignant peripheral nerve sheath tumor (MPNST) is challenging, especially in cases without neurofibromatosis 1 (NF1), because MPNST lacks specific markers on immunohistochemistry (IHC). METHODS: We performed IHC for histone 3 trimethylated on lysine 27 (H3K27me3) and evaluated the percentage of cells with H3K27me3 loss using measured values at 10% intervals, categorized as complete loss (100% of tumor cells lost staining), partial loss (10% to 90% of tumor cells lost staining), and intact (no tumor cells lost staining). We conducted fluorescence in situ hybridization (FISH) for NF1 and p16 deletions comparing 55 MPNSTs and 35 non-MPNSTs, consisting of 9 synovial sarcomas (SSs), 8 leiomyosarcomas (LMSs), 10 myxofibrosarcomas (MFSs), and 8 undifferentiated pleomorphic sarcomas (UPSs). We assessed the percentage of cells with homozygous and heterozygous deletions and defined "deletion" if the percentage of either the NF1 or p16 deletion signals was greater than 50% of tumor cells. RESULTS: Among the 55 MPNSTs, 23 (42%) showed complete H3K27me3 loss and 32 (58%) exhibited partial loss or intact. One each of the 9 SSs (11%), 8 LMSs (12%), and 8 UPSs (12%) showed complete H3K27me3 loss and many non-MPNSTs exhibited intact or partial H3K27me3 loss. Among the 55 MPNSTs, 33 (60%) and 44 (80%) showed NF1 or p16 deletion, respectively. Co-deletion of NF1 and p16 was observed in 29 (53%) MPNSTs. Among the 23 MPNTSs showing H3K27me3 complete loss, 18 (78%) and 20 (87%) exhibited NF1 or p16 deletion, respectively. Among the 32 MPNSTs with H3K27me3 partial loss or intact, 15 (47%) and 24 (75%) exhibited NF1 or p16 deletion, respectively. The frequency of NF1 and/or p16 deletion tended to be lower in non-MPNSTs than in MPNSTs. Approximately 90% of MPNSTs included cases with H3K27me3 complete loss and cases showing H3K27me3 partial loss or intact with NF1 and/or p16 deletion. Approximately 50% of MPNSTs showed co-deletion of NF1 and p16 regardless of H3K27me3 loss. CONCLUSIONS: FISH for NF1 and p16 deletions, frequently observed in high-grade MPNSTs, might be a useful ancillary diagnostic tool for differentiating MPNST from other mimicking spindle cell and pleomorphic sarcomas.

Clinicopathological and prognostic significance of H3K27 methylation status in malignant peripheral nerve sheath tumor: correlation with skeletal muscle differentiation.

Malignant peripheral nerve sheath tumor (MPNST) is a very aggressive peripheral nerve sheath-derived sarcoma, which is one of the most difficult tumors to diagnose due to its wide spectrum of histological findings and lack of specific immunohistochemical markers. Recently, it has been reported that losses of expression of H3K27me3 and H3K27me2 caused by PRC2 dysfunction may be useful diagnostic markers for MPNST, but there is no consensus on their clinicopathological significance. Here, we investigated the relationship between loss of H3K27 methylation and various parameters and clarified the clinicopathological significance of such loss. We analyzed the clinicopathological and immunohistochemical features in 84 MPNST cases. Complete losses of H3K27me3 and H3K27me2 were observed in 37 (44%) and 29 (35%) cases, respectively. Losses of H3K27me3 and H3K27me2 were significantly correlated with myogenic immunopositivity (H3K27me3 vs. desmin, P = 0.0051; H3K27me3 vs. myogenin, P = 0.0009; H3K27me2 vs. myogenin, P = 0.042). Meanwhile, there were significant correlations between preservation of immunohistochemical neurogenic markers and intact H3K27me3 and H3K27me2 (H3K27me3 vs. S-100 protein, P = 0.0019; H3K27me3 vs. SOX10, P = 0.014; H3K27me2 vs. S-100 protein, P = 0.0011; H3K27me2 vs. SOX10, P = 0.0087). In multivariate analysis, local recurrence, distant metastasis, high FNCLCC grade, and loss of SOX10 expression were independent prognostic factors for overall survival. H3K27me3 and H3K27me2 expression was retained in all 26 cases of rhabdomyosarcoma non-alveolar subtype. In conclusion, we suggest that H3K27me3 and H3K27me2 immunonegativity is useful but not definitive for diagnosing MPNST. Complete loss of H3K27 methylation may be involved in aggressive transdifferentiation from neural differentiation to skeletal muscle differentiation in MPNST.

EZH2 expression is associated with inferior overall survival in mantle cell lymphoma.

Enhancer of zeste homolog 2 (EZH2) is a catalytic component of the polycomb repressive complex 2 (PRC2) which reduces gene expression via trimethylation of a lysine residue of histone 3 (H3K27me3). Expression of EZH2 has not been assessed systematically in mantle cell lymphoma (MCL). Expression of EZH2 was assessed by immunohistochemistry in 166 patients with MCL. We also assessed other PRC2 components and H3K27me3. Fifty-seven (38%) of MCL patients were positive for EZH2 using 40% cutoff. EZH2 expression was associated with aggressive histologic variants (65% vs. 29%, p < 0.001), high Ki-67 proliferation rate (median, 72% vs. 19%, p < 0.001), and p53 overexpression (43% vs. 2%, p < 0.001). EZH2 expression did not correlate with expression of other PRC2 components (EED and SUZ12), H3K27me3, MHC-I, and MHC-II. Patients with EZH2 expression (EZH2+) had a poorer overall survival (OS) compared with patients without EZH2 expression (EZH2-) (median OS: 3.9 years versus 9.4 years, respectively, p < 0.001). EZH2 expression also predicted a poorer prognosis in MCL patients with classic histology (median OS, 4.6 years for EZH2+ and 9.6 years for EZH2-negative, respectively, p < 0.001) as well as aggressive histology (median OS, 3.7 years for EZH2+ and 7.9 years for EZH2-negative, respectively, p = 0.046). However, EZH2 expression did not independently correlate with overall survival in a multivariate analysis. Gene expression analysis and pathway enrichment analysis demonstrated a significant enrichment in cell cycle and mitotic transition pathways in MCL with EZH2 expression. EZH2 expression detected by immunohistochemistry is present in 38% of MCL cases and it is associated with high proliferation rate, p53 overexpression, aggressive histologic variants, and poorer OS. Based on gene expression profiling data, EZH2 expression could potentiate cell cycle machinery in MCL. These data suggest that assessment of EZH2 expression could be useful to stratify MCL patients into low- and high-risk groups.